The development of the various hematopoietic cell lineages is compartmentalized during fetal development and throughout adult life. At approximately day 12 of embryonic development, the fetal liver (FL) is seeded by definitive hematopoietic stem cells, which arise from the aorta-gonad-mesonephros region of the developing embryo (Cumano and Godin, 2001). The FL continues as the primary site of hematopoietic development until birth, when the bone marrow (BM) takes over as the primary site for hematopoiesis in the adult.
The process of stem cell differentiation is tightly regulated by soluble factors and cell contact-dependent signals within specialized microenvironments, each of which support the development of specific cell lineages. During lymphopoiesis, the thymic environment is required for the differentiation of hematopoietic progenitor cells (HPCs) into T lymphocytes (Anderson et al., 1996). On the other hand, B cell development takes place within the BM microenvironment (Osmond, 1994). The process of hematopoiesis can be modeled in vitro using BM-derived stromal cell lines (Dorshkind, 1990). A number of BM derived stromal cell lines have been developed in recent years, which are capable of supporting the development of multiple hematopoietic cell lineages. For instance, the OP9 BM stromal cell line (Kodama et al., 1994) has been shown to support the differentiation of HPCs into multiple lineages, including B cells, in vitro (Carlyle et al., 1997). However, efforts to generate T cells from HPCs in vitro in the absence of a thymic microenvironment have been unsuccessful. This is thought to be due to unknown factors acting at multiple developmental stages within the three-dimensional architecture of the thymus (Anderson et al., 1996; Lind et al., 2001).
The process of T cell development from HPC to mature TCR-αβ+ T cell consists of a series of commitment events and multiple developmental checkpoints, including TCR V(D)J gene rearrangement,TCR-β-selection, and positive/negative selection of developing thymocytes. Efforts to recapitulate one or more of these events in vitro have had to depend on the use of fetal thymic organ culture (Anderson et al., 1996). The molecular interactions responsible for this thymus dependency remain largely unknown. However, a number of recent studies have implicated Notch receptor-ligand interactions in the earliest T cell lineage commitment events (MacDonald et al., 2001). Notch signaling is an evolutionarily conserved pathway that controls multiple cell fate decisions throughout ontogeny. Notch signaling is initiated by the local interaction of Notch receptors with Notch ligands on neighboring cells (Artavanis-Tsakonas et al., 1999). In vertebrates, these ligands consist of Jagged and Delta-like family members. Engagement of the Notch receptor results in its proteolytic cleavage by a presenilin dependant γ-secretase activity (Taniguchi et al., 2002), followed by the translocation of the cleaved intracellular domain of Notch to the nucleus, where it binds to CBF-1/RBP-Jκ and activates transcription of downstream target genes (Artavanis-Tsakonas et al., 1999).
Several lines of evidence implicate Notch signaling at various stages of lymphocyte development. Specifically, it has been suggested that Notch signaling promotes TCR-αβ+ T cell development at the expense of TCR-γδ+ T cell development (Washburn et al., 1997). Furthermore, a number of investigators have proposed various roles for Notch in the development of CD4+ and CD8+ single positive (SP) T cells from CD4+ CD8+ (DP) precursor thymocytes (Deftos et al., 2000; Izon et al., 2001; Robey et al., 1996; Wolfer et al., 2001). There is also data from studies that address the role of Notch signaling in governing T cell versus B cell fate decisions by lymphocyte progenitors. Specifically, B cell development is abolished in mice reconstituted with BM progenitors expressing a constitutively active form of Notch, rather DP T cells develop in the BM of these mice (Pui et al., 1999). In a complementary experiment, Notch-1 conditionally-deficient mice show a severe block in T cell development, with the concomitant development of B cells in the thymus (Radtke et al., 1999). These results strongly support the notion that Notch signaling is critical for the earliest stages of T cell commitment. A further role of Notch signals at early stages of T cell development has recently begun to be elucidated. Radtke and colleagues demonstrated that a conditional inactivation Notch-1 at the CD44+ CD25+ (DN2) stage of T cell development results in a partial block at the subsequent CD44− CD25+ (DN3) stage (Wolfer et al., 2002). This was shown to be the result of inefficient V to DJ recombination at the TCR-β locus. In contrast, rearrangements at the TCR-γδ locus were not affected.
The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.